Pdf (Ac- Cessed 31.01.2008) This Study Was Funded Through the EC (MODELKEY De Lange H.J., De Jonge J., Den Besten P.J., Oosterbaan J

Biologia 67/1: 180—199, 2012 Section Zoology DOI: 10.2478/s11756-011-0158-3

Theresponseofmacroinvertebratecommunitytaxa and functional groups to pollution along a heavily impacted river in Central Europe (Bílina River, Czech Republic)

Claus Orendt1,GeorgWolfram2, Zdeněk Adámek3,PavelJurajda3 & Mechthild Schmitt-Jansen4

1Orendt-Hydrobiologie WaterBioAssessment, Brandvorwerkstr. 66, 04275 Leipzig, Germany; e-mail: [email protected] 2DWS Hydro-Okologie,¨ Zentagasse 47, 1050 Vienna, Austria; e-mail: [email protected] 3Department of Fish Ecology, Institute of Vertebrate Biology, Academy of Sciences of the Czech Republic, Kvetna 8,CZ- 60365 Brno, Czech Republic; e-mails: [email protected], [email protected] 4Department of Bioanalytical Ecotoxicology Helmholtz-Centre for Environmental Research – UFZ, Permoserstr. 15, 04318 Leipzig, Germany; e-mail: [email protected]

Abstract: Macroinvertebrate communities were investigated along a gradient of heavy industrial and municipal pollution in the highland Bílina River (Czech Republic). Physico-chemical determinants and ions were monitored and community analysis performed focusing on taxonomic composition, ecological functioning (feeder and dweller guilds) and water quality metrics, including saprobity index, BMWP and diversity. Impacted sites differed significantly from reference and from recovered stretches. Chemical data revealed two main pollution factors, (1) a “salinity determinant”, described best by conductivity 2− − and SO4 , and (2) an “organic pollution determinant”, represented best by O2 concentrations and NO2 , all varying locally and temporally. Some metrics and taxa showed significant correlations to abiotic parameters. Functional communities showed a stronger relationship to the “organic pollution determinant”, suggesting that elevated organic pollution had a dominating influence on functional community metrics; though other variables may also have an influence in this multi- stress environment. On the other hand, there were indications that the taxonomic community was more influenced by ion concentrations (“salinity determinant”). The gradient from reference sites to polluted sites was weaker in the final sampling campaign. The results presented here can be used as a reference for assessing future changes in environmental impact from pollution, being finer and more detailed than assessment according to the EU’s WFD. Key words: community analysis; functional community; taxonomic community; multiple pollution; multi-stress; macroinvertebrates; Central Europe; lower mountain river; EU-WFD

Introduction fer from multiple stressors (Tockner et al. 2010), which may confound successful implementation of the Euro- Changes in water quality along a river’s course affect pean Union’s Water Framework Directive (EU-WFD; not only taxonomic composition but also the function EC 2000). In addition to saprobity, chemical pollution of a community. Numerous studies have used macroin- and loss of habitat may also contribute to changes, or vertebrates in order to trace the sources and extent even a decline, in macroinvertebrate assemblages. This of pollution or to monitor the degradation or recov- has already been observed in the Bílina and Elbe rivers ery of a stream. In the Western Palearctic and Nearc- (Adámek & Jurajda 2001; Adámek et al. 2010) and has tic bioregions, traits and tolerances of macroinverte- to be taken into consideration when searching for the brate taxa are sufficiently well known to allow appro- key causes of ecological degradation. In the St. Clair priate bioindication levels to be applied (Griffiths 1991; River in Canada, Griffiths (1991), among others, found Orendt 1999; D’Surney et al. 2000; Timm et al. 2001; that physical habitat characteristics and sediment con- Statzner et al. 2005; Berenzen et al. 2005; Gupta & taminants explained different kinds of macroinverte- Sharma 2005; de Deckere et al. 2007). One of the most brate communities and their numbers. Moreover, the frequently used tools for measuring biological water influence of pollution on the taxonomic community and quality is the index of saprobity, which reflects pollu- on ecological function depends on its concentration, tion by organic, decomposable wastewaters. This was which may have different effects on different taxa. De developed >100 years ago (Kolkwitz & Marsson 1902), Lange et al. (2004), in their studies on the Rhine-Meuse and later extended and refined by Zelinka & Marvan delta, found that moderate levels of contamination af- (1961), Sládeček (1973), Mauch et al. (1985) and others. fected the structure, but not the productivity, of the Nowadays, there is increasing awareness that rivers suf- benthic macroinvertebrate community. Thus, when a

c 2011 Institute of Zoology, Slovak Academy of Sciences Response of macroinvertebrates to river pollution 181 water body is to be restored, it is in the interests of water managers to determine exactly which factors are E N, responsible for the degradation in order to fulﬁl the re- 78 quirements of the EU-WFD. The Bílina River, a tribu- ◦ + + + 23.39 0.2 13 138

tary of the Elbe River in the North-West of the Czech 2 39 ◦ Republic, is considered one of the most polluted run- ◦ ning waters in the country. According to Jurajda et al. E14 N, 50

(2010), the river is at high risk of failing the require- ments of the WFD. Our knowledge about the biota of · – the Bílina River Basin is, however, quite limited. Re- + + 21.81 175 N39.26 18.5

cently, Jurajda et al. (2010) released the results of a 36 53 ◦ ﬁrst survey of macroinvertebrate and ﬁsh assemblages ◦ at locations along the main channel and some small E13 N, 50

tributaries. The aims of this study were to (1) investigate · 28.69 – + + 23.66 206 the macroinvertebrate communities of this heavily im- 31.1 51 36 pacted river along the pollution gradient in the main ◦ ◦ channel, and (2) to elaborate the factors responsible

for the response of the community to general pollution E13 N, 50 along the stream. An analysis of organochemical compounds will be treated separately in a following publi- 49.2 54.53 + + + 216 45.1

cation. 30 41 Obrnice Chotějovice Velvety Ústí n.L. ◦ ◦ N, 50

Study area and sample sites E13

The Bílina River (Fig. 1), a tributary of the Elbe River, is 1.86 + + + 55.46 238 49.3 Most located in the North-West of the Czech Republic and has a 39 30 2 ◦ catchment area of 1,070.9 km . It arises from a spring in the ◦ Krušné hory Mts (785 m a.s.l.) and, after 84.2 km, the river ﬂows into the Elbe River at Ústí nad Labem (132 m a.s.l.). N, 50 E13 Its course is interrupted by two reservoirs (river km 72.2,

km 66.8) and diversion piping (km 60.4–57.4). The river is 3.09 ++++++ + + + 31.57 244 53.2

considered as the most polluted stream in the Czech Repub- 37 32 ◦ lic, having received pollutants from heavy industry, brown ◦ coal mining, energy and chemical industries, and municipal wastewater treatment plants (WWTPs) for more than 100 E13 N, 50 years.

Samples were taken from eight locations along the · 51.23 + + + – 47.43 251 river’s course (Fig. 1, Table 1). We avoided following a to- 59.0 33 31 ◦ pographical gradient along the river so as not to hinder in- ◦ terpretation of the impact of chemical compounds. All sampling sites were similar in terms of hydromorphological char-

acter and belong to the same potamal stream zone, while E13 N, 50 data on aquatic communities presented by Jurajda et al. (2010) show a relatively high morphological homogeneity of 3393 22.53 + + + + 285 river zone. Morphological variation between the study sites 65.2 30 29 ◦ ◦ was low and evaluation of morphological feature data did unpolluted unpolluted polluted polluted polluted polluted polluted polluted 13 50 not show a significant gradient, indicating no major influ- Kyjicka reservoir ence on community distribution that could mask the direct Jirkov, downstream Komořany Litvínov response of biota the direct response of the biota to water quality. Further, there was no significant difference in pH from neutral values (7.47 ± 0.26 SD) between sites, suggesting that this system is well buffered by the geochemical background or organic pollutants. Mobilisation of toxic 22–24 Oct. 2007 22–24 Apr. 2008 heavy metals due to acidity, therefore, does not appear to 19–22 June 2006 play a major role in pollution of the Bílina River. The highest upstream site (km 65.2) is located downstream of a reservoir. Between this site and that following (km 59.0), the river is diverted through piping for three kilometres. Since these two sites are located upstream of the Site name Stream km m a.s.l. Coordinates Pollution* first major inflow of wastewater, they are considered as ‘ref- Sampling date 24–27 Sep. 2007 erence’ sites for sites situated downstream. These sites do Table 1. Sampling site and event characteristics. Explanations: + sampling performed, – no sampling, *) Pre-classification according results from CzHI (2011). not represent true reference sites, as required by the WFD, 182 C. Orendt et al.

Fig. 1. Positions (according to river km) of the eight sample sites along the Bílina River (see also Table 1). Arrows indicate waste water treatment plants. however, as low levels of pollution are still received from Stockinger (2005), Orendt (2008) and a reference collection. within the catchment area, as seen from monitoring data of The numbers of individual taxa were counted and classified the local water agencies (CzHI 2011). At km 54.0, upstream in abundance classes according to AQEM protocols (AQEM of the site at km 53.2, outlets discharge from the Litvínov 2004). town WWTP and the Chemopetrol Litvínov Co. refinery, Physico-chemical data were measured at the same time while some kilometres downstream (before the site at km as macroinvertebrate sampling (Table 2) using electronic 49.3), there is a large WWTP serving the town of Most. instrumentation (WTW Ltd.). One-litre samples of water Further downstream, industrial and wastewater inflow de- were taken on each sampling date for ion analysis, the wa- creases along the whole stretch up to the town of Ústí nad ter being kept cool during transport and then deep-frozen Labem (km 0.2). until chemical analysis. Inductively coupled plasma atomic emission spectroscopy (SpectroCiros CCD) following EN ISO 11885 (1998) was used for the analysis of Ca2+,Mg2+, + 4+ Na ,Si2 and total Fe, while ion chromatography (Dionex Material and methods 3− − − − 2− DX500) was applied for PO4 ,NO3 ,NO2 ,Cl and SO4 . Field work Sampling took place over four campaigns from 2006 to Data analysis and statistics 2008 (Table 1). Macroinvertebrates were collected by kick Taxa abundance was estimated into seven classes (1 = sin- sampling (500 µm mesh size) according to EN 27828:1994. gle record to 7 = highly abundant) according to categories All mesohabitats (e.g., large cobbles, boulders, submerged suggested by DIN 38410 (2004). Both metrics and ecological macrophytes, debris) from both lotic and lentic habitats cov- quality classes according to the WFD were calculated using ering >5% of the substrate surface within a 100 m stretch the ecological running water assessment software tool in AS- were merged in the sample in order to avoid the effects of lo- TERICS V.3.1 (www.fliessgewaesserbewertung.de; AQEM cal variability on ecological traits and biotic indices (Brabec 2004; University of Duisburg-Essen 2008). Where statisti- et al. 2004; Sychra et al. 2010). At each site, one three- cal analysis and metric calculation required abundance data minute sample was taken. All material was sieved (500 µm (e.g. distribution of feeding types, dweller types, stream type mesh size) and transferred to one or two 1 L bottles for profiles using categories defined by Illies & Botosaneanu transport to the laboratory. A solution of 37% formalde- 1963), a transformation of the class values into numeri- hyde was added to obtain a final concentration of approxi- cal values was performed as recommended for the proce- mately 5% for preservation. The relative abundance of eas- dure in ASTERICS. Additional metrics, such as species ily identifiable taxa was noted in the field. In the labora- richness, number of EPT (Ephemeroptera, Plecoptera, Tri- tory, macroinvertebrates were identified to the lowest possi- choptera) taxa, BMWP (Biological Monitoring working ble taxonomical level using keys by Sperber (1950), Wachs party; Hellawell 1978) and the Czech Index of Saprobity (1967), Brinkhurst (1971), Wiederholm (1983, 1986), Moller (SI; CSN 75 7716, 1998), were calculated for description Pillot (1984a, 1984b), Rivosecchi (1984), Pitsch (1993), of community structure and organic pollution. A Principle Sauter (1995), Gittenberger et al. (1998), Glöer & Meier- Component Analysis (PCA; based on a variance-covariance Brook (2003), Vallenduuk (1999), Vallenduuk & Moller Pil- matrix) was performed in order to reduce the large dataset lot (1999), Klink (2002), Killeen et al. (2004), Neu & To- and to highlight important physico-chemical variables (us- bias (2004), Bauernfeind & Humpesch (2001), Glöer (2002), ing scores from the first two factors as criteria). Data were Hölzel (2002), Janeček (2003), Langton & Visser (2003), log-transformed in order to avoid dominant influences of Timm & Veldhuijzen van Zanten (2003), Eiseler (2005), Wil- variables with high values. Correspondence analysis (CA) son & Ruse (2005), Lechthaler & Car (2005), Lechthaler & of the taxonomic community was undertaken in order to Response of macroinvertebrates to river pollution 183 ) ∗ 0.2 not categorized ) ∗ 18.5 not categorized ) ∗ 31.1 not categorized ) ∗ 45.1 impacted not categorized 49.3 impacted 53.2 reference 59.0 reference 34444334 6.6 6.07–8.643.4 7.5 2.987–3.82.8 7.27–10.33 3.6 16.8 2.8–4.1 2.237–4.397 14.63–19.57 4.6 21.5 9.5 3.373–5.23 18.19–21.82 35.6 4.5–16.7 4.3 34.01–36.25 17.9 3.773–5.2 26.8 22.37–31.15 4.7–54.4 5.2 27.7 19.81–29.43 14.2 4.611–5.64 30.3 4.8 0.1–44.6 29.4–31.3 15.7 4.74–4.956 5.6 9.5–38.9 4.273–6.06 38.5 5.0 24.0–53.1 3.578–6.3 25.0 22.2–37.4 27.9 22.6–36.3 7.3 6.9–7.7 7.4 7.3–8.0 7.4 7.3–7.6 7.5 7.0–7.8 7.6 7.4–7.9 7.3 7.3–7.5 7.5 7.08–7.6 7.6 7.5–7.9 235 204–274 305 260.5–369 774 540–1395 835 515–1307 1020 734–1234 898 628–1160 891 602–961 923 607–964 10.8 10.7–10.821.0 10.5 20.4–21.6 6.31–11.017.0 23.3 10.7 13.9–20.1 22.2–25.40.20 22.1 6.2–11.3 47.7 0.20–0.35 20.5–26.70.06 0.24 36.1–52.949.8 6.5 101.4 0.05–0.17 0.22–0.2916.1 56.8 59.4–142.8 49.2–50.3 0.180.19 2.4–8.6 125.6 0.23 14.7–17.4 59.2 33.3–61.4 0.06–0.19 0.19–0.19 55.5–147.7 14.3 0.17–0.25 54.3–73.6 113.2 76.9 7.6 0.87 13.35–15.5 0.26 192.8 0.20 59.8–141.8 56.3–83.5 0.28–1.35 0.16–0.39 130.3–240.5 52.6 2.2–10.6 70.6 216.9 0.16–0.43 60.2 0.69 0.37 32.2–91.2 108.0–281.7 54.5–86.7 4.9 0.23 290.0 0.41–3.65 0.15–0.51 67.8 180.1–309.4 52–68.4 81.1 0.17–0.24 0.17–7.2 180.2 1.77 0.36 36.7–90.0 140.3–220.1 70.0 49.2–101.8 0.15 197.9 0.66–2.8 0.16–0.4 66.0 91.5 8.5 51.7–80.4 134.7–240.9 0.14–0.17 221.2 37.7–104.5 46.0–105.5 0.89 79.8 0.52 134.0–237.5 0.15 46.2 6–9.2 0.28–1.5 0.32–0.62 0.14–0.22 39.9–52.4 56–85.5 0.69 9.5 0.51 0.19 53.2 0.45–0.93 0.23–1.38 0.14–0.21 33.3–58.4 6.3–10.2 0.45 0.17 60.1 0.32–1.04 0.13–0.39 37.1–73.5 0.63 0.17–1.03 65.2 median min–max median min–max median min–max median min–max median min–max median min–max median min–max median min–max 1 1 1 1 1 1 1 1 1 1 1 1 − − − − − − − − − − − − S µ C 11.7 8.3–15.7 9.9 9.2–13.7 14.1 10.7–24.2 13.6 10.2–22.2 20.2 17.5–22.8 13.4 8.8–23.1 12.7 8.7–22.7 12.6 8.1–23.7 ◦ mg L mg L mg L mg L mg L mg L mg L mg L mg L mg L − − − 3 − 2 3 4 2+ 2 4 + 2+ (see Table 1) − 4+ 2 Pollution category pH Ca Mg Si NO SO Cl PO Na tot FeNO mg L Table 2. Selected Physico-chemical parameters and ionStream measurements. km N Temperature *) But, pollution assumed. Conductivity cm O 184 C. Orendt et al.

Fig. 2. Plot of sample sites (different dates) according to field measurements and ion concentrations using the first two components of a PCA (1st component: 57.6% of total variance; 2nd component 16.6%; 3rd component: 8.6% [not included in the plot]) showing geographical distribution. Explanation of codes: 59-1 = sampling site river km 59.0 on the first campaign (June 2006), 59-2 = sampling site river km 59.0 on the second campaign (September 2007). etc. A – Different symbols represent reference, impacted and non-categorised sites, respectively. Arrow: upstream to downstream, illustrating local variation. + = polluted sites, reference sites, • site km 45.1 and 31.1. B – Different symbols represent the different sampling dates of the four campaigns. Arrow: direction of first to last sampling campaign, illustrating seasonal variation. April 2008, × October 2007, • September 2007, + June 2006. Response of macroinvertebrates to river pollution 185

Fig. 3. Conductivity along the study reach over four separate campaigns (June 2006, Sept 2007, Oct 2007, Apr 2008). Explanation of codes: 59-1 = sampling site river km 59.0 on the ﬁrst campaign (June 2006), 59-2 = sampling site river km 59.0 on the second campaign (September 2007), etc.

find relevant taxa and to reduce the taxa list, thereby allow- Table 3. Field measurement and ion concentration loading for the ing observation of further relationships with environmental PCA. variables. Canonical Correspondence Analysis (CCA), using Component untransformed data, was performed in order to study relationships between biological metrics and abiotic parameters 1234 from sampling sites during different sampling campaigns. Spurious correlations between environmental variables were Variance (%) 57.6 16.6 8.6 7.0 traced by applying partial correlation analysis. The Kruskal- 2− Wallis test was used to identify significant differences be- SO4 0.946 0.241 0.132 –0.040 tween metrics, variables and sites, while the Mann-Whitney Conductivity 0.898 0.330 0.081 –0.151 Ca2+ 0.895 0.018 0.361 0.183 test was applied for detailed pair-wise tests between single 2+ sample sites. The Wilcoxon Signed Rank test was used to Mg 0.870 0.026 0.300 0.249 Cl− 0.830 0.231 0.260 –0.0892 search for differences between dates at the sample sites, and Na+ 0.824 0.430 0.135 –0.210 Spearman’s rank correlation (1-tailed) was used to test for − NO2 0.205 0.912 0.130 0.032 bivariate correlation. PCA, partial correlations calculated O2 –0.317 –0.730 –0.409 0.192 3− with SPSS, CCA, CA, and statistical tests were calculated PO4 0.366 0.108 0.843 –0.160 − using the PAST software package (version 2.07, 2011; Ham- NO3 0.188 0.380 0.797 0.199 4+ mer et al. 2001) Si 0.321 –0.052 –0.085 0.871 Total Fe 0.356 0.034 –0.094 –0.839

Results

Physico-chemical characteristics and ions variance), suggesting that the inflow of inorganic loads An overview of the data measured is given in Table 2. at km 53.2 is the main contributor to pollution on the PCA revealed both geographical and temporal differ- river. Nitrite and oxygen contributed most to the sec- ences in the data. The geographical gradient is repre- ond factor; however, this explains far less of the variance sented by a clear separation in the position of the ref- (16.6%) than the first factor. The third contribution erence sites (km 65.0, 59.0) from impacted sites (km factor, which contained relatively high levels of phos- 53.2, 49.3) during all four sampling dates (Fig. 2A). phate and nitrate, explained a relatively small level of Differences between the impacted sites and sites down- variance (8.3%). The first and strongest PCA factor stream (km 45.1 to km 0.2) are less striking, indicat- can be summarised as a “salinity” factor, while the sec- ing similar environments. Temporal variation is illus- ond and third factors are “organic pollution” factors, trated by a second PCA perspective (Fig. 2B), which contributing less to variance in the sample dataset and shows a steady unidirectional change from the first to having a lower gradient. The longitudinal distribution the last sampling date for all sites (shown as an exam- of the compounds and parameters measured shows a ple in Fig. 3). The scores (Table 3) identify sulphate, clear increase in concentrations of almost all chemical conductivity, and a number of other ions as principle parameters from reference to impacted sites. Conduc- contributors to the first factor (55.4% of cumulative tivity, an indicator of mineral load, was highest down- 186 C. Orendt et al.

Fig. 4. A–C. Taxonomic metrics for the investigated sites along the Bílina River (in river km). Each dot represents the value of one campaign at a given site. D – Abundance of Chironomus riparius (see methods section for classes) on different sampling dates. stream of the km 49.3 impacted site (Fig. 3), while cal- tem is well buffered by the geochemical background or cium, magnesia and sulphate all showed a further inorganic pollutants. Thus, mobilization of toxic heavy crease at the km 53.2 and km 49.3 impacted sites. This metals due to acidity does not play a major role in the pattern was observed during all campaigns, as revealed pollution, here. by the PCA plot (Fig. 2). Interestingly, elevated values of phosphate were also found at the reference sites dur- Metrics and response of selected taxa along the river ing the first two campaigns. The data for phosphate, stretch however, varied greatly over time and, in April 2008, A total of 309 taxa were recorded during the four sam- values for phosphate at the reference sites were also pling campaigns (see Appendix 1). An assessment of the lowest observed. Oxygen concentrations were higher ecological status derived from the macroinvertebrate at the first impacted site (km 53.2) than at the refer- community indicated little variation between sampling ence sites upstream, but dropped towards the second sites: classes 3 (moderate) or 4 (poor) for all samples. impacted site at km 49.3 (Table 2). Measurements of The impacted sites (km 53.2 and 49.3) did not differ temperature were excluded from the multivariate eval- substantially from either the reference or other sites. uation as the values were generally dependant on season Only between reference site km 59.0 and the final site and, therefore, not useful for further analysis. A sepa- (km 0.2) was a “moderate” status reached. rate analysis, however, showed small but significantly Taxa richness (Fig. 4A) dropped remarkably be- higher values at the impacted sites compared to the tween reference site km 59.0 and the downstream im- km 65.0 reference site on each sampling date. At km pacted sites at km 53.2 and 49.3, narrowly missing sta- 18.5, significantly lower temperature values were ob- tistical significance (P < 0.051; Mann-Whitney; n = served than at km 45.1, and significantly higher values 29). Compared to the other sites, taxa numbers at km than at the km 65.0 reference site (P < 0.05; Wilcoxon 53.2 showed great variation (from 18 in September 2007 test). to 61 in April 2008). Taxa richness at the second im- pH did not differ much around neutral values (7.47 pacted site (km 49.3) showed less variability and dif- ± 0.26 SD) between the sites suggesting that this sys- fered significantly only from the final station at km 0.2. Response of macroinvertebrates to river pollution 187

Fig. 5. Assessment and functional metrics for the investigated sites along the Bílina River (in river km). Each dot represents the value of one campaign at a given site.

The difference to reference km 59.0 was close to signifi- Table 4. Functional group (guild) loadings of the first two PCA cance (P < 0.051, Mann-Whitney). At km 45.1, higher components from all dates and sites (initial data: dominances taxa richness values were regularly recorded (median = in %). 55). Downstream, richness dropped (median = 41) but Component increased again to higher values towards the mouth of the river at km 0.2 (median = 62). 12 Total EPT abundance (Fig. 4B) clearly followed Explained variance (%) 81.5 11.6 the same pattern (P < 0.001; Kruskal-Wallis), while oligochaete abundance generally showed the opposite Metric pattern (P < 0.003; Kruskal-Wallis; Fig. 4C). Differ- ences in oligochaete abundance between reference site Gatherers/Collectors 227.20 20.64 Grazers/Scrapers 97.88 –25.04 km 65.2 and km 0.2, and between the impacted sites at Predators 73.90 –26.06 km 53.2 and 49.3, were significant (P < 0.030; Mann- Passive filter feeders 32.00 –17.58 Whitney). The difference between reference site km Active filter feeders 28.68 –1.07 59.0 and the impacted sites was close to significance Shredders 18.04 –1.11 (P < 0.051; Mann-Whitney). A similar distribution was Phytal dwellers 158.70 –46.39 found for Chironomus riparius, whichisanindicatorfor Lithal dwellers 106.65 –40.28 pollution (Fig. 4D). This species was very abundant at Pelal dwellers 95.34 21.66 Psammal dwellers 73.34 12.81 impacted sites km 53.2 and km 49.3, but occurred only Akal dwellers 45.06 –1.09 scattered at km 45.1 (P < 0.014; Kruskal-Wallis). On the last sampling date, however, only low abundance was recorded and then only at site km 53.2. Water quality measurements varied significantly 81.5%, 2nd component: 11.6%; Table 4). Aside from along the study stretch. SI (Fig. 5A) increased from ref- phytal dwellers and grazer/scrapers, all guilds men- erence site km 59.0 to impacted km 53.2 (and further tioned showed significant changes in community domi- downstream of that site), though a significant differ- nance (P < 0.05; Kruskal-Wallis; n=28; pelal and lithal ence was only recorded between reference site km 65.0 dwellers are shown as examples in Fig. 5). and polluted site km 45.1 (P < 0.030; Mann-Whitney). When taxa were used for PCA instead of metrics BMWP values (Fig. 5B) showed clearer significant dif- (using only taxa with a frequency <3 and abundance ferences between sites than SI (P < 0.004; Kruskal- <5, resulting in around 100 taxa), total variance of the Wallis). The difference between both reference sites and 1st component was much lower and little difference was the second polluted site at km 49.3 were also significant observed between components (1st component: 15.7%; (both P < 0.043; Mann-Whitney), as was the drop from 2nd component: 12.6%; 3rd component: 10.3%), indicat- the first polluted site at km 53.2 to the second at km ing a weak gradient in the data. Nevertheless, the three 49.3 (P < 0.037; Mann-Whitney). groups (references sites, polluted sites and others) were Gatherers/collectors, grazers/scrapers and preda- still clearly separated (plot not shown). tors (feeding guilds), and phytal, lithal and pelal Summing up, in comparison with the reference dwellers (habitat guilds) explained most of the variance sites at km 65.2 and km 59.0, both the taxonomic and in the first component of PCA analysis (1st component: functional community changed significantly following 188 C. Orendt et al.

show the relationships between biological metrics and physico-chemical parameters and ion concentrations. 2− The CCA dataset consisted of (1) conductivity, SO4 , − 3− O2 concentrations, NO2 and PO4 as environmental variables (indicated as important by PCA analysis; see Table 3), (2) those metrics significantly correlated to abiotic parameters (P < 0.05; Spearman rank correlation), and a reduced taxa list containing only taxa with a frequency >2 in all samples and a score >0.7 in a preceding CCA including all taxa. The CCA (Fig. 6) revealed a strong affinity between EPT abundance, BMWP score, shares of hypocrenal dwellers, and number of taxa of different levels to O2 concentrations. Pelal dweller, gatherer/collector dominance and oligochaete abundance − were plotted close to the NO2 variable. The shares of Oligochaeta and Diptera in relation to total taxa abundance had an affinity to the “salinity” determinant (de- 2− scribed by SO4 and conductivity). The SI score was 3− close to PO4 concentrations. The first axis of the CCA scores explained 90.57% of total variance, which is very high. A plot of sample sites did not reveal any grouping Fig. 6. Plot of CCA axis scores (axis 1: 90.56% of explained pattern, whether by season or position along the stretch variance; axis 2: 4.97%; axis 3: 3.23%) using metrics. Abbre- (figure not shown). viations: BMWP – BMWP score; epirhithral – share (percent- When taxa were used for the CCA instead of met- age) of epirhithral habitat type in the community; EPT abund – total abundance of all Ephemeroptera, Plecoptera, Trichoptera rics, the first axis explained only 40.51% of total vari- taxa; Gath Coll – share (percentage) of gatherer and collector ance, the second 32.03%, and the third 20.03%. The feeding type in the community; hypocr – share (percentage) of triplot (Fig. 7), however, indicates a clear grouping of hypocrenal habitat type in the community; N Fam – number of reference, impacted and non-categorised sites. The ref- families; N Gen – number of genera; N Taxa – number of (high resolved) taxa; Oligo Abund – total abundance of Oligochaeta; erence sites were connected to the “organic load de- Oligo+Dipt/totaltaxa – share of abundances of (Oligochaeta + terminant” in the plot, being associated with the O2 − Diptera)/abundance of all taxa; Pel – share (percentage) of pelal determinant and opposite to the NO2 . Some samples habitat type in the community; Shredd – share (percentage) of from km 18.5 and site 0.2 were also close to this group. shredder feeding type in the community; SI Cz – Czech index of saprobity; SI Zel Mar – index of saprobity according Zelinka & A further group comprising the two sites downstream of Marvan. the impacted sites (km 45.1 and km 31.1) was linked to the “salinity determinant”, represented by high values 2− inflow of chemical industry wastewater at km 53.2. Eco- of conductivity and SO4 . The clearly separated group logical functions changed slightly downstream of the of impacted sites was situated close to the “organic load heavily impacted sites, indicating a level of recovery, determinant”, also indicated by its opposition to the 3− but conditions declined again after km 41.8, only in- PO4 variable. creasing once again at the end of the river (km 0.2). At Taxa distribution according to site (Fig. 7) in- this latter site, the distribution of ecological functions dicates that C. riparius was strongly linked to poor (i.e., feeding or dwelling guild, taxonomic diversity and environmental conditions at the polluted sites, while SI) were similar to those at reference sites km 65.2 and Hydropsyche angustipennis and Glyptotendipes pallens km 59.0, upstream of the pollution source at Litvínov (both filter feeders) showed a preference for the refer- (km 53.2). ence sites, not being recorded at other sites. Scores for the first axis of the CA, while significantly correlated − Relationships between metrics and environmental vari- to NO2 (rS = 0.402; P < 0.04) showed a somewhat ables stronger correlation to conductivity (rS = 0.674; P < Though many significant correlations were observed be- 0.001), suggesting that the taxonomic community re- tween metrics and abiotic variables, among metrics, and flected the ion concentration (“salinity determinant”) with SI when bivariate tests were used, a partial cor- gradient better than the “organic determinant” (though relation procedure indicated that most were spurious, its contribution should not be neglected). − only that between pelal dweller dominance and NO2 proving to be truly significant. Due to autocorrelations Discussion and crosswise factor influences in the dataset, multivariate treatments proved most useful for studying the Abiotic features relationships between environmental variables and met- Analysis of physico-chemical parameters and ion con- rics or taxa. For this reason, a CCA (Fig. 6) was used to centrations revealed both temporal and local gradients. Response of macroinvertebrates to river pollution 189

Fig. 7. Plot of CCA axis scores (axis 1: 40.5% of explained variance; axis 2: 32.1%; axis 3: 20.0%) using taxa (taxlist reduced). polluted sites, reference sites, • site km 45.1 and 31.1, ◦ site km 18.5 and 0.2. Aﬂ – Ancylus ﬂuviatilis,Afu–Anabolia furcata,Ahe – Alboglossiphonia heteroclita,Alo–Ablabesmyia longistyla,Bfu–Baetis fuscatus,Bte–Bithynia tentaculata,Cdi–Chaetogaster diaphanus,Cri–Chironomus riparius,Csp–Chironomus sp., Dic – Dicrotendipes sp., Dsp – Diamesa sp., Gco – Glossiphonia complanata,Gly–Glyptotendipes sp., Gpa – Glyptotendipes pallens,Han–Hydropsyche angustipennis,Hsp–Haliplus sp., Hst – Helobdella stagnalis,Hyd–Hydra sp., Lul – Lumbriculidae gen. sp., Nbi – Neureclipsis bimaculata,Nbr–Nais bretscheri,Ort– Orthocladiinae gen. sp., Pgs – Psychodidae gen. sp., Ppe – Paratanytarsus penicillatus,Seq–Simulium equinum,Sfe–Spirosperma ferox,Sss–Simulium (Simulium)sp.,Sws–Simulium (Wilhelmia) sp., Tas – Tanypodinae gen. sp., Vpi – Valvata piscinalis.

Both conductivity and other ions followed this pat- Biotic metrics and biota tern, at least concerning changes in water quality at Our data were in harmony with previous findings in lit- the impacted sites. Levels for O2 and ions of N and erature, i.e. that a number of frequently used metrics, P compounds did not increase downstream, however, even one as simple as taxa richness, show significant but decreased towards the end of the study stretch. As responses to pollution and recovery. Resh et al. (2000) with conductivity, both compound load and differences has stated that richness metrics were most accurate in tended to decline between the first and last sampling detecting impairment, while Lenat & Penrose (1996) campaign, suggesting a general reduction in pollution have shown that EPT taxa richness is even more sta- after the inlet and from the first to last sampling date. ble and predictable than total taxa richness. Compared Our results suggest two main pollution sources, one de- to these, BMWP, being based on macroinvertebrate riving from “salinity” loading and the other from or- family level, is likely to be less sensitive to ecosystem − ganic loading. The organic pollution comprised NO2 , change than taxa richness metrics at the species level, − 3− − NO3 and PO4 variables, in addition to O2,withNO2 although the former showed a clear response, in our ions and O2 showing higher statistical importance than study. − 3− NO3 and PO4 (PCA) and a stronger significant cor- Of the functional metrics used, only correlation of − − − − relation for O2 with NO2 than NO3 .AsNO3 may NO2 and pelal dwellers proved to be positively signif- − have originated from mineral wastewater and NO2 is icant. High pelal dweller dominance occurred at pol- + a decomposition product of NH4 derived from organic luted sites, and on some dates, but was also comparable − wastewater, we used NO2 as the descriptor for organic to that at the reference sites during the last sampling pollution during further evaluation. campaign, when these sites were least polluted. This Our data also indicated that the reference sites suggests that habitat distribution of pelal dwellers may were affected by a certain amount of organic pollution. also be connected to the level of pollution rather than to Due to the relatively low number of sampling dates, locally consistent morphological structures alone, i.e., however, we were unable to smooth extreme values and high organic load can result in increased organic sedi- outliers and, therefore, all physico-chemical data should mentation. Moreover, the distribution of lithal dweller be interpreted with care. The data were, however, suf- dominance was also significant; suggesting that there ficient to allow elaboration of plausible interpretations may have been more morphological differences than the of the relationships between physico-chemical and bio- protocols used in this investigation were capable of de- logical data. tecting. 190 C. Orendt et al.

While all metrics used reflected levels of organic however, conductivity levels were >2foldhigherthan pollution adequately, no dramatic differences in SI were in our study, suggesting that only highly tolerant taxa noted over the stretch investigated, though levels were were to be found in the waters studied. Other authors statistically significant. For example, SI confirmed find- have, however, reported clear effects (e.g., Crowther & ings from chemical measurement and indicated that the Hynes 1977; Dickman & Gnochauer 1978; Demers & reference sites were also affected by organic pollution. Sage 1990). In an early study, Turoboyski (1959; cited − The stronger correlation of SI with NO2 rather than in Necchi Júnior et al. 1994) studied pollution caused other abiotic variables (rS = 0.703; P < 0.001) suggests by salinity and organic matter, though using microbial that the functional community was responding directly communities. More recent studies have monitored the to an “organic pollution determinant” rather than a complex pollution patterns caused by industrial activ- “salinity” determinant. The taxonomic community, on ities, both with and without municipal sewage run-off the other hand, showed a somewhat stronger relation- (Mooraki et al. 2009; Arimoro et al. 2011). In these ship to the “salinity determinant”, as indicated by a studies, however, differentiation between “salinity” load − stronger correlation to conductivity than to NO2 .This and organic wastewater was either not detected or not suggests that the taxonomic community may better analysed for. Tho et al. (2006) were able to elaborate reflect the “salinity determinant” than the functional separate influences from shrimp monoculture (pollution community. Taxonomic composition and abundance de- resulting in eu- to hyperhaline salinity) and human in- termine the functional community in the analysis, how- fluence (organic pollution), but did not discuss indi- ever, whereas its functioning may be more complex. cation by macroinvertebrates. Whereas Braukmann & Dominance of C. riparius, for example, at polluted loca- Böhme (2011) were able to show clear changes in the tions, e.g. km 53.2, indicates not only low competition taxonomic macroinvertebrate community due to indus- caused by a lack of other species (indirect indication) trial salt contamination (rather than to other types of but has also been found to be an effect of growth stim- chemical variable or organic pollution) in the Werra ulation caused by the combined appearance of both or- River (Germany), variation and level were not marked. ganic and toxic pollution (direct indication), as found Once again, however, comparison with our results is by Stuijfzand et al. (1996) in studies from the Meuse limited as ecological function was not analysed for and River. maximum chloride concentrations were around 20-times The fact that the taxonomic community and func- higher and maximum conductivity 6-times higher than tional metrics respond differently to different types in our study. of environmental pressure has also been addressed by In contrast to the above, our results showed that other authors, though with differing results. Pinel- the “organic pollution determinant” had a stronger ef- Alloul et al. (1996) found that taxonomic and func- fect on the functional community than the taxonomic tional community indices followed a pollution gradi- (though this does not mean that there was no taxo- ent indicated by chemical compounds in different ways. nomic community response). Further, CCA also indi- Moreover, both indices stressed different aspects of cated that certain taxa were clearly related with the − macroinvertebrate community structure, with a clear organic pollution parameters O2 and NO2 .Withless response to morphology. Further, during investigation organic pollution, however, the “salinity pollution de- of macroinvertebrate communities from differing mor- terminant” was better reflected by the taxonomic com- phological areas (lentic and lotic), Brabec et al. (2004) munity. These results may be a result of the particular has shown that assessment results differ under the same character of the study stretch and reflect the unique level of pollution. While Simiao-Ferreira et al. (2009) pattern of pollution. found a response to pollution that was clearly caused only by sewage; it required low variation in morpho- References vs. impacted sites logical features. In our study, we also found no sub- As indicated by the SI and BMWP values above, the stantial morphological differences between the sites in- first reference site appears to have been somewhat de- vestigated, strongly suggesting that pollution was the graded by chemical pollution. This may have been main influence on community structure. This finding is caused by the outflow of higher amounts of organic ma- similar to that of Adámek & Jurajda (2001). terial and other chemical and metabolic compounds at We found no similar reference in the literature to site km 65.2 than at the next site downstream. The the degree in which the “salinty factor” and “organic higher SI value at km 65.2 this caused must make the factor” were indicated by the taxonomic and functional use of this site as a true “reference” questionable. Re- communities, respectively, in this study. However, Pis- garding dweller type dominance, total taxa number, cart et al. (2005) did observe a decrease in macroinver- EPT taxa number, SI and BMWP, the site was eco- tebrate taxonomic richness in a small stream affected logically similar to those near the river’s mouth at sites by a salinity gradient and, in terms of ecological func- km 18.5 and km 0.2. In consequence, the site at km 59.0 tion, there followed a slight change in the relative abun- is the only true reference site for this study. dance of invertebrate feeding groups. Blasius & Merritt A range of metrics showed highest values at the (2002), when investigating the effects of road salt on km 53.2 and km 49.3 impacted sites. At the latter site, stream communities, found no substantial impact on ei- the results at the end of the study period (April 2008) ther taxonomic or functional community. In their study, were less different from the other sites than in the previ- Response of macroinvertebrates to river pollution 191 ous sampling dates, suggesting reduced pollution dur- AQEM 2004. 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rivers: an initial assessment of trait patterns in least im- Vallenduuk H.J. & Moller-Pillot H.K.M. 1999. Key to the lar- pacted river reaches. Freshwater Biol. 50 (12): 2136–2161. vae of Chironomus in Western Europe. RIZA Rapport Vol. DOI: 10.1111/j.1365-2427.2005.01447.x No/97.053,Lelystad,38pp. Stuijfzand S.C., Shen D., Helms M. & Kraak M.H.S. 1996. Effects Vallenduuk H.J. 1999. Key to the Larvae of Glyptotendipes Kief- of pollution in the river Meuse on the midge Chironomus fer (Diptera, Chironomidae) in Western Europe. RIZA rap- riparius. Proc. Exp. Appl. Entomol. N.E.V. Amsterdam 7: port Vol.No/97.052, Lelystad, 46 pp. 211–216. Wachs B. 1967. Die häufigsten hämoglobinführenden Oligochae- Sychra J., Adamek Z. & Petrivalska L. 2010. Distribution and ten der mitteleuropäischen Binnengewässer [Common central diversity of littoral macroinvertebrates within extensive reed European oligochaets with haemoglobin]. Hydrobiologia 30 beds of a lowland pond. Ann. Limnol. – Int. J. Limnol. 46 (2): 225–247. (4): 281–289. DOI:10.1051/limn/2010026 Wiederholm T. (ed.) 1983. Chironomidae of the Holarctic Region. Tho N., Vromant N., Nguyen T.H. & Hens L. 2006.Organic Keys and Diagnosis. Part 1. Larvae. Entomologica Scandi- pollution and salt intrusion in Cai Nuoc District, Ca Mau navica Supplementum 19: 1–457. Province, Vietnam. Water Environ. Res.78 (7): 716–723. Wiederholm T. (ed.) 1986. Chironomidae of the Holarctic region. DOI: 10.2175/106143006X101755 Keys and diagnosis. Part 2. Pupae. Entomologica Scandinav- Timm H., Ivask M. & Möls T. 2001. Response of macroinverte- ica Supplementum 28: 1–482. brates and water quality to long-term decrease in organic pol- Wilson R.S. & Ruse L.P. 2005. A guide to the identification lution in some Estonian streams during 1990–1998. Hydrobi- of genera of chironomid pupal exuviae occurring in Britain ologia 464 (1–3): 153–164. DOI: 10.1023/A:1013999403209 and Ireland (including common genera from northern Eu- Timm T. & Veldhuijzen van Zanten H.H. 2003. Freshwater rope) and their use in monitoring lotic and lentic fresh wa- Oligochaeta of North-West Europe. CD. http://www.eti.uva. ters. Freshwater Biological Association Special Publications nl ISBN: 9075000448 13, 176 pp. ISBN: 978-0-900386-73-2 Tockner K., Pusch M., Borchardt D. & Lorang M.S. 2010. Zelinka M. & Marvan P. 1961. Zur Präzisierung der biologischen Multiple stressors in coupled river-floodplain ecosystems. Klassifikation der Reinheit fließender Gewässer [Refining the Freshwater Biol. 55 (1): 135–151. DOI: 10.1111/j.1365- biological classification of the purity of running waters]. Arch. 2427.2009.02371.x Hydrobiol. 57 (3): 389–407. University of Duisburg-Essen 2008. ASTERICS V3.01, http:// www.fliessgewaesserbewertung.de/en/download/berechnung Received June 5, 2011 (accessed 20.11.2009) Accepted November 6, 2011 194 C. Orendt et al.

Appendix 1. List of taxa recorded and their abundance in classes according to DIN 38410 (2004): Abundance class 1 = 1–2 specimens per sample (single to scattered); abundance class 2 = 3–10 (sparse to several); abundance class 3 = 11–30 (medium density); abundance class 4 = 30–100 (fairly dense); abundance class 5 = 101–300 (abundant, dense); abundance class 6 = 301–1000 (very abundant, very dense); abundance class 7 = >1001 (heaps). Reference sites in bold, polluted sites in italics. Abbreviations: grp. – group; Ad. – adult; Lv. – larva.

Date 2006-06-20 2006-06-20 2006-06-20 2006-06-21 2006-06-21 2006-06-21 2007-09-24 2007-09-24 2007-09-25 2007-09-25 2007-09-24 2007-09-24 2007-09-24 2007-10-24 2007-10-22 2007-10-22 2007-10-24 2007-10-23 2007-10-23 2007-10-23 2007-10-23 2008-04-23 2008-04-23 2008-04-23 2008-04-22 2008-04-23 2008-04-22 2008-04-22 2008-04-22

Site (stream km) 65.2 59.0 53.2 49.3 45.1 0.2 65.2 53.2 49.3 45.1 31.1 18.5 0.2 65.2 59.0 53.2 49.3 45.1 31.1 18.1 0.2 65.2 59.0 53.2 49.3 45.1 31.1 18.5 0.2

Hydrozoa Hydra sp. 7343424 ..551.... 1 2 ...1 . 433312 Turbellaria Dendrocoelum lacteum (O.F. Mueller, 1774) ...... 11...... 2 ...... Dugesia lugubris (Schmidt, 1861) ..... 2 ...... Dugesia lugubris/polychroa 4 . ..2 ..... 14...... 233..... 2 .. Dugesia sp...... 2 ....1 ...... 33 Dugesia tigrina (Girard, 1850) ...... 1 ...... Polycelis tenuis Ijima, 1884 ...... 1 ...... Polycelis sp...... 1 ...... Turbellaria Gen. sp...... 2 ..... Nematoda Nematoda Gen. sp. . 1 23. 1 2 . 1 . 2 . 2 11..222. 2 . 2 . 1321 Bivalvia Anodonta sp...... 1 .. Musculium lacustre (O.F. Mueller, 1774) ....1 ...... 1 .. Pisidium casertanum casertanum (Poli, 1791) ...... 2 . Pisidium casertanum ssp. (Poli, 1791) ...... 2 ...... 2 ...... Pisidium henslowanum (Sheppard, 1823) ...... 2 ...... 3 ...... Pisidium sp. . 2 2 . 113 ... 12. 52... 23114....21 Pisidium subtruncatum Malm, 1855 ...... 1 . 2 . ....32...... Sphaerium corneum (L., 1758) . 3 ...... 2 ...... 3 ...... 2 . Sphaerium sp...... 12.. Gastropoda Acroloxus lacustris (L., 1758) ...... 1 .. Ancylus ﬂuviatilis O.F. Mueller, 1774 . 1 ... 1 . ....12. 4 .....3 ...... 1 Bithynia tentaculata (L., 1758) . 2 ..22. ..1 . 4 ...... 12....11. 1 Gyraulus albus (O.F. Mueller, 1774) ..1 . ..2 ..1 . 2 . 2 . ..1 ....2 ...... Physella acuta Draparnaud, 1805 ... 5 2 ..1 . 4 ...... 3 .....35.... Physella sp...... 252 ...... 2 ... Physidae Gen. sp. ..3 ...... Planorbidae Gen. sp. ....1 ...... Potamopyrgus antipodarum (J.E. Gray, 1843) ...... 1 ..1 . 1 ..2 ...... Radix auricularia (L., 1758) ..1 ...... Radix balthica (L., 1758) ...... 4 ...... 2 ...... Radix labiata (Rossmaessler, 1835) ...... 1 ...... Radix sp...... 2 ...... Valvata piscinalis ssp. (O.F. Mueller, 1774) ..2 . 2 ....4 ...... 3 ....1 ...... Hirudinea Alboglossiphonia heteroclita (L., 1758) ...... 5 ...... 4 . 1 Erpobdella nigricollis (Brandes, 1899) ...... 13..... 233 Erpobdella octoculata (L., 1758) . 2 ..545 ..234562..242533..1644 Erpobdella sp...... 22.....2 Erpobdella vilnensis (Liskiewizcz, 1925) ...... 1 . Erpobdellidae Gen. sp. 1 ...242 ..222...... 42...2 . 2 ... Glossiphonia complanata (L., 1758) ...... 2 ..1 . ... 211... 1 14.. Glossiphonia concolor (Apathy, 1888) ...... 1 Glossiphonia sp...... 2 ...... 2 . 1

Glossiphoniidae Gen. sp...... 2 ......

12 Helobdella stagnalis (L., 1758) . 1 2 . 525 ¾ 75324 . ..74422. 6521 Hemiclepsis marginata (O.F. Mueller, 1774) ..... 1 ...... Theromyzon tessulatum (O. F. Mueller, 1774) ...... 1 ...... 1 ...... Oligochaeta Aulodrilus pluriseta (Piguet, 1906) ...... 1 ...... Chaetogaster diaphanus (Gruithuisen, 1828) . 2 433 . 3 ..3 ...... 331 ... Eiseniella tetraedra (Savigny, 1826) ...... 1 .

Enchytraeidae Gen. sp. ..2 ..3 ...... 12..2 ..

32 32 Limnodrilus claparedeanus Ratzel, 1868 ..323 ..¿ 42.... 334..3 23.. Response of macroinvertebrates to river pollution 195

Appendix 1. (continued)

Site (stream km) 65.2 59.0 53.2 49.3 45.1 0.2 65.2 53.2 49.3 45.1 31.1 18.5 0.2 65.2 59.0 53.2 49.3 45.1 31.1 18.1 0.2 65.2 59.0 53.2 49.3 45.1 31.1 18.5 0.2

43 3 Limnodrilus hoﬀmeisteri Claparede, 1862 . 4 51472 431353 433. 36 . 3554 Limnodrilus sp. ... 3 ...... Lumbricidae Gen. sp. 1 ....1 ...... 2 ...... 11....21 Lumbriculidae Gen. sp. ..232...... 3 ....1 . 2 ..... Lumbriculus variegatus ... 5 2 ...... 5 ...... 1532.. Nais barbata (O.F. Mueller, 1773) ...... 2 ..14...... 22. 15. 3442.. Nais bretscheri Michaelsen, 1899 ...... 2 ...... 2 Nais communis Piguet, 1906 ...... 2 ...... Nais elinguis O.F. Mueller, 1773 ...... 4 ...... 3 ..5532. 5 Nais pardalis Piguet, 1906 ...... 2 ...... Nais pseudobtusa Piguet, 1906 . 2 ...... 2 Nais simplex Piguet, 1906 ...... ½ 32...... 3 ...... Nais sp...... 2 ...... Nais variabilis Piguet, 1906 ...... 1 ...... 6 Ophidonais serpentina (O.F. Mueller, 1773) . 3 434 ... ½ 24...... 46..54 . 54. 3

Potamothrix hammoniensis (Michaelsen, 1901) ...... ¾ ...1 ...... 1 . 1 .....2 Potamothrix moldaviensis (Vejdovsky et Mrazek, 1902) ...... 2 ...... 1 Pristina rosea (Piguet, 1902) ...... 1 ...... Psammoryctides albicola (Michaelsen, 1901) ...... 3 ... 43...... 14. Psammoryctides barbatus (Grube, 1861) ..... 2 . ... 444..... 235..... 554 Rhyacodrilus sp. ... 2 ...... Slavina appendiculata (D’Udekem, 1855) ...... 1 ...... Spirosperma ferox (Eisen, 1879) ...... 1 ... 43...... 3 ..1 . ... 221 Stylaria lacustris (L., 1767) . 6 555 . 7 ..134233. 6 66315. 6767. 6

Tubifex tubifex (O.F. Mueller, 1773) ..3 . . 3 ...... 1 . 4 ...421441

66 Tubiﬁcidae Gen. sp. ...3 ..2 544555 6 . 6545433665 Crustacea 11 2 Asellus aquaticus (Linnaeus sensu Racovitza, 1919) . 4 24766 . ¿ 767764 656744 . 5477 Gammarus fossarum/pulex ...... 1 .. Acari Hydrachnidia Gen. sp. 2 . 22121 ..2 ..4 ...... 3 . 11. 1 ..4 Plecoptera Nemoura sp. ..1 ...... Isoperla sp...... 1 . Odonata Calopteryx sp...... 1 ...... Calopteryx splendens (Harris, 1782) 1 ...... 1 ...... 2 ...... Coenagrionidae Gen. sp. ....1 ...... Platycnemis pennipes (Pallas, 1771) . 1 ...... 1 ...... Zygoptera Gen. sp...... 1 ..... Ephemeroptera Baetis buceratus Eaton, 1870 ...... 4 .

Baetis fuscatus (L., 1761) . 2 1 . . 2 2 ½ . ...5 . 2 ...... Baetis rhodani (Pictet, 1843) ..... 1 ...... 3345. 146 Baetis sp. 132 . 244 ....24. 6 ..2 . 27. 1 2 . ..23 Baetis vernus Curtis, 1834 . 6 4 . 465 ... 255. 4 ..3 . 54....21. 7 Caenis horaria (L., 1758) ...... 2 ...... Caenis luctuosa (Burmeister, 1839) . 1 2 . ..1 ...... 1 .....1 Caenis sp...... 1 . 1 ...... Centroptilum luteolum (Mueller, 1776) ...... 2 ..1 ....2 ...... Potamanthus luteus (L., 1767) ...... 1 ...... Serratella ignita (Poda, 1761) ..... 1 ...... Trichoptera Anabolia furcata . 1 ...... 3 ....11 Anabolia nervosa (Curtis, 1834) . 1 ...... 2 ...... Athripsodes cinereus (Curtis, 1834) . 2 ...... 1 . ... 1 ...... Athripsodes sp...... 2 ...... Goera pilosa (F., 1775) . 2 ...... 1 ...... Halesus digitatus/tesselatus ...... 3 ...... Hydropsyche angustipennis ssp. (Curtis, 1834) . 3 ....7 ..114. 63..1 . 2 . 62..2 . 1 . Hydropsyche contubernalis ssp. Mclachlan, 1865 ..... 2 . ....14...... 5 ...... 3 Hydropsyche incognita Pitsch, 1993 ...... 1 196 C. Orendt et al.

Appendix 1. (continued)

Site (stream km) 65.2 59.0 53.2 49.3 45.1 0.2 65.2 53.2 49.3 45.1 31.1 18.5 0.2 65.2 59.0 53.2 49.3 45.1 31.1 18.1 0.2 65.2 59.0 53.2 49.3 45.1 31.1 18.5 0.2

Hydropsyche pellucidula (Curtis, 1834) ...... 2 Hydropsyche pellucidula grp...... 2 ...... Hydropsyche sp...... 2 6 .....5 57....2542....23 Hydroptila sp. . 1 ..21. ....1 ..1 .....122..5 ..5 Hydroptilidae Gen. sp...... 1 ...... Leptoceridae Gen. sp...... 2 ...... 2 ...... Limnephilidae Gen. sp...... 4 ...... Limnephilus sp...... 1 ...... Molanna angustata Curtis, 1834 ...... 1 ...... Mystacides nigra (L., 1758) ...... 2 ...... Neureclipsis bimaculata (L., 1758) ...... 4 ...... 42...... 31...... Psychomyia pusilla (F., 1781) ...... 2 . 2 .....2 ...... 4 Tinodes sp. ..1 ...... Lepidoptera Lepidoptera Gen. sp...... 1 ...... Megaloptera Sialis lutaria (L., 1758) ...... 1 ...... Heteroptera Corixinae Gen. sp...... 1 ...... Corixini Gen. sp...... 1 ..... Micronecta sp...... 2 .....2 . 1 ...... Gerris lacustris (L., 1758) ...... 1 ...... Coleoptera Elmis maugetii Latreille, 1798 ...... 1 ...... Elmis sp...... 11. ..1 ... Elmis sp. Ad...... 1 Haliplus ﬂuviatilis Aube, 1836 ....1 ...... Haliplus immaculatus Ad. Gerhardt, 1877 ...... 1 .. Haliplus sp. ....1 ....142...... 211..... 11. Hydraena sp. Ad...... 1 ...... Limnius volckmari (Panzer, 1793) . 1 ...... Orectochilus villosus (O.F. Mueller, 1776) ...... 3 ...... 2 ...... Platambus maculatus Ad. (L., 1758) ...... 1 ...... Platambus maculatus Lv. (L., 1758) ...... 2 ...... Diptera – Simuliidae Prosimulium sp...... 1 ...... Simulium (Nevermannia) sp. ..1 ...... 1 ...... Simulium (Simulium)sp...... 2 ...... 1 1

Simulium (Simulium) sp...... ¾ . . 455... . 4 ...1 . .... ½ Simulium (Wilhelmia) sp...... 2 . ¾ 1 . 542 . ....2 ...... Simulium angustipes Edwards, 1915 ..... 1 ...... Simulium equinum (L., 1758) ...... 54...... 2

Simulium erythrocephalum (DeGeer, 1776) ..222 . 6 ¾ . 256343..143143..2232 Simulium lineatum (Meigen, 1804) ...... 22...... Simulium morsitans Edwards, 1915 ...... 2 ...... 2 Simulium ornatum Meigen, 18 18 ..4 . ...¾ . ..52...... 1 . 21. 1 . 12 Simulium ornatum Meigen, 1818 ...... 1 ...... Simulium reptans (L., 1758) ..2 . . 4 ...... 2 ...... Simulium sp. ..4 . . 4 4 ...... 23..2 . 62...... Diptera – Chironomidae Ablabesmyia longistyla Fittkau, 1962 ... 2 2 ...... 2 . .... Brillia bifida Kieffer, 1909 ...... 2 . 2 . .... Brillia flavifrons (Johannsen, 1905) . 2 ...... 2 . 2 . 2 . 21 Brillia sp...... 2 Cardiocladius capucinus (Zetterstedt, 1850) ...... 22. 2 ...... Cardiocladius fuscus Kieffer, 1924 . 3 ... 2 ...... 2 ... Chaetocladius piger (Goetghebuer, 1913) ...... 3 . . 2 .. Chironomini Gen. sp...... 2 ...... Chironomus nuditarsis Keyl, 1961 ...... 1 ...... 1 .. Chironomus nudiventris Ryser, Scholl et Wuelker, 1983 ...... 3 . 1 . Chironomus plumosus (L., 1758) ...... 1 ... Chironomus plumosus grp. ....4 ...... Response of macroinvertebrates to river pollution 197

Appendix 1. (continued)

Site (stream km) 65.2 59.0 53.2 49.3 45.1 0.2 65.2 53.2 49.3 45.1 31.1 18.5 0.2 65.2 59.0 53.2 49.3 45.1 31.1 18.1 0.2 65.2 59.0 53.2 49.3 45.1 31.1 18.5 0.2

77 42 Chironomus riparius Meigen, 1804 ..77... .....1 21...... 1 . Chironomus sp. ... 1 6 ....42...... 122.....222. Clinotanypus nervosus (Meigen, 1818) ...... 2 . .... 2 Conchapelopia sp. ..43...½ . ....47..2 ...5 . . . 233 Conchapelopia melanops (Meigen, 1818) ...... 3 4 . 32. 2

Conchapelopia grp. ..6 ...... ¾

Orthocladiini COP 1 ...42. ¾ . 223...... 2 . 74256246

55 Cricotopus (Cricotopus) sp. . 5 ....6 4622. 7 751...... Cricotopus (Isocladius) sp. . 2 ...... 56 45 Cricotopus bicinctus (Meigen, 1818) . 5 5 . 64. ½ . 132. 22 644254 5443 Cricotopus albiforceps (Kieffer, 1916) . 5 4 ...... 1 ...... 322 . 211. Cricotopus fuscus (Kieffer, 1909) . 2 4 . . 3 ...... 1 1 . 1 ...21543232 Cricotopus sp. . 3 ...... Cricotopus sp. 7 . 55. 7 ...... 1 1 . . 1 . 176632547 Cricotopus sylvestris (F., 1794) . 2 . 1 4 ...... 1 ...... 1 . 2 21.. Cricotopus tremulus (L., 1758) ...... 2 ..1 . Cricotopus trifascia Edwards, 1929 ..... 2 ...... 1 ...... Cryptochironomus obreptans grp...... 2 ...... Cryptochironomus sp...... 2 ...... 11. 2 ...... Cryptotendipes pseudotener (Goetghebuer, 1922) . 2 ...... Diamesa kasymovi/tonsa ...... 2 . . 1 .... Diamesa sp...... 5222. 2 . 2 Diamesa tonsa (Haliday, 1856) ...... 3 . .... Dicrotendipes pulsus (Walker, 1856) ...... 3 ...... Dicrotendipes nervosus (Staeger, 1839) ..3772. ..462134..352. 533 . 6 . 4 . Dicrotendipes sp...... 1 ....4 . 3 ...... Eukiefferiella brevicalcar (Kieffer, 1911) ...... 1 ...... 3 . 4 ... Eukiefferiella claripennis (Lundbeck, 1898) . 4 ..42...... 1 12223.... 5 3311 Eukiefferiella clypeata (Kieffer, 1923) ...... 2 ...... 1 Eukiefferiella fuldensis Lehmann, 1972 . 2 ... 2 ...... Eukiefferiella ilkleyensis (Edwards, 1929) ..... 2 ...... Eukiefferiella sp...... 2 . 1 . ..21..... 12. 2346..22 Glyptotendipes barbipes (Staeger, 1839) ...... Glyptotendipes ospeli Contreras-Lichtenberg ....3 ...... et Kiknadze, 2000 Glyptotendipes pallens (Meigen, 1804) . 3 ....7 ...... Glyptotendipes paripes (Edwards, 1929) . 3 ..3 . 2 ..56..7 . ..23..52..24. 1 Glyptotendipes sp...... 4 .....1 ... 1 2 ...6 . ..2 ... Harnischia curtilamellata (Malloch, 1915) . 2 ..3 ...... Harnischia fuscimanus Kieffer, 1921 . 3 ...... Harnischia sp. . 1 ...... 3 ...... Heterotrissocladius marcidus (Walker, 1956) ..... 1 ...... Limnophyes sp...... 1 ...... Macropelopia sp. ....2 ...... Micropsectra atrofasciata (Kieffer, 1911) . 3 5 . 53...... 1 . 314362213 . 2311 Micropsectra sp. ..43. 4 . ..4 . 7 . 243444722. 777777 Microtendipes pedellus (De Geer, 1776) . 3 ...... 2 4 . . 132 Microtendipes pedellus grp. . 2 ..252 ..2422. 3 ..1123...... Nanocladius dichromus (Kieffer, 1906) . 5 4161. ..2 ..2 2 . . 1 411222. 1 2351 Nanocladius dichromus grp...... 23...... Nanocladius rectinervis (Kieffer, 1911) ..... 1 ...... Nanocladius sp...... 2 ...... 1 ...... Orthocladiinae Gen. sp. 2 ...... 2 25...... Orthocladius (Euorthocladius) sp...... 1 ...... Orthocladius ashei Soponis, 1990 ...... 1 ...... 233211 Orthocladius glabripennis (Goetghebuer, 1921) ...... 1 . 2 . 31.. Orthocladius lignicola Kieffer, 1914 ...... 2 Orthocladius luteipes Goetghebuer, 1938 ...... 1 .... Orthocladius oblidens (Walker, 1856) ...... 1 ...... Orthocladius obumbratus Johannsen, 1905 ...... 1 . 2 . .... Orthocladius olivaceus (Kieffer, 1911) ...... 1 ...... Orthocladius pedestris Kieffer, 1909 ...... 2 ...... 198 C. Orendt et al.

Appendix 1. (continued)

Site (stream km) 65.2 59.0 53.2 49.3 45.1 0.2 65.2 53.2 49.3 45.1 31.1 18.5 0.2 65.2 59.0 53.2 49.3 45.1 31.1 18.1 0.2 65.2 59.0 53.2 49.3 45.1 31.1 18.5 0.2

Orthocladius rivicola grp...... 2 . . 3 2 ..2 Orthocladius rubicundus (Meigen, 1818) ...... 1 .....12. ....12 Orthocladius sp...... 1 ....1 . ..1 ... Orthocladius thienemanni Kieffer, 1906 ...... 1 ..2121 Parachironomus gracilior (Kieffer, 1918) . 4 ...... 2 . .... Parachironomus gracilior grp. . 1 ....2 ...... 1 ...... 22. Parachironomus vitiosus grp...... 2 ...... Paracladius conversus (Walker, 1856) ... 1 4 ...... Paracladopelma nigritulum (Goetghebuer, 1942) ... 1 ...... Parametriocnemus stylatus (Spaerck, 1923) ...... 1 ...... Paratanytarsus dissimilis Johannsen, 1905 . 5 3 . 3 ..... 21..1 ... 2 . 123412344 Paratanytarsus penicillatus (Goetghebuer, 1928) ...... 2 22. 242 Paratanytarsus sp. 174 . . 2 . ... 62337..26. 557....76 Paratendipes albimanus (Meigen, 1818) . 2 ...... Paratendipes albimanus grp. . 2 ..44...... 2 ... 27..4 2 . . 4 .. Paratrichocladius rufiventris (Meigen, 1830) ....42...... 2 . 131 Pentaneurini Gen. sp...... 2 ...... Phaenopsectra sp. ..32. 2 ...... 1 ....1 ..2 . 4 ..2223 Polypedilum bicrenatum Kieffer, 1921 ..2 ...... Polypedilum convictum (Walker, 1856) ...... 2 ...... Polypedilum cultellatum Goetghebuer, 1931 ..... 2 ...... 2 ...

Polypedilum laetum (Meigen, 1818) ...... ½ ...... Polypedilum nubeculosum (Meigen, 1804) . 2 2 . 52. ..22..23..2 ....4 ...... 43 54 Polypedilum pedestre (Meigen, 1830) ..72. 2 . . ½ .....2 ...1 .. 2221 Polypedilum scalaenum/pullum . 2 ... 5 ...... 2 . 2 ....76...... Polypedilum scalaenum grp...... 1 ...... Polypedilum sp...... 1 ...... 122. Polypedilum tritum (Walker, 1856) ...... 1 ... Potthastia gaedii (Meigen, 1838) ...... 1 Potthastia gaedii grp. 42...... Potthastia longimanus Kieﬀer, 1922 . 4 ...... 1 ...... 2 ...... Potthastia longimanus grp...... 2 ...... 12...... Procladius (Holotanypus) sp. ... 2 2 ...... 1 ....3 ...... Procladius choreus (Meigen, 1804) ...... 2 ...... Prodiamesa olivacea (Meigen, 1818) ..2 . 32...... 12. 144 . 4 . 1 . Rheocricotopus atripes (Kieﬀer, 1913) ...... 1 ..1 ...... Rheocricotopus atripes grp. . 3 4 . 55...... 1 . 2 ...... 67 Rheocricotopus chalybeatus (Edwards, 1929) ...... 1 2132

Rheocricotopus effusus (Walker, 1856) ...... ¾ ...... 1 ...... 53 Rheocricotopus fuscipes (Kieffer, 1909) ..42. 5 5 ¾ . ..5 ...... 264 322. Rheocricotopus sp...... 12. 2 ..1 ...... Rheopelopia ornata (Meigen, 1838) . 2 ...... Rheopelopia sp. . 4 ....2 ...... Rheotanytarsus pentapoda (Kieffer, 1909) ....72...... Rheotanytarsus photophilus (Goetghebuer, 1921) . 3 ...... 1 ...... 122 Rheotanytarsus sp. ....5 . 4 ....2 ...... Saetheria reissi Jackson, 1977 ...... 3 ... Synorthocladius semivirens (Kieffer, 1909) . 4 ... 3 ...... 2 ...... 12. 222111 Tanypodinae Gen. sp...... 2 ...... 1 ....5 . 1 .... Tanytarsus sp...... 2 ...... 3 ...... Thienemannia sp...... 2 .. Tvetenia calvescens (Edwards, 1929) . 3 2 . 34. ....5 . 141 . 11411221. 111 Tvetenia sp...... 5 ...... 4 ...... 12...... 6 Tvetenia verralli (Edwards, 1929) . 3 ... 3 ...... 1 ...... 2 .....2 Tvetenia discoloripes/verralli ...... 2 ...... Xenochironomus xenolabis (Kieffer, 1916) ...... 2 Diptera – other families Atrichops crassipes (Meigen, 1820) ...... 2 ...... Bezzia grp. 1 . ..12. ..1 . 21. 2 1 . ...3 ...... Bezzia sp...... 121 . 1 ..4 Ceratopogonidae Gen. sp...... 1 ...... Chelifera sp...... 2 ...... Response of macroinvertebrates to river pollution 199

Appendix 1. (continued)

Site (stream km) 65.2 59.0 53.2 49.3 45.1 0.2 65.2 53.2 49.3 45.1 31.1 18.5 0.2 65.2 59.0 53.2 49.3 45.1 31.1 18.1 0.2 65.2 59.0 53.2 49.3 45.1 31.1 18.5 0.2

Culex sp. ..1 ...... Dicranomyia sp...... 1 ...... Dicranota sp. 23...... Diptera Gen. sp. ....2 ...... 1 . . 1 11...... Dolichopodidae Gen. sp...... 1 .... Empididae Gen. sp. ..1 ...... 1 ..1 .. Ephydridae Gen. sp...... 1 ... Hemerodromia sp...... 1 ...... 1 . ..12 Limnophora sp...... 2 ...... Limoniidae Gen. sp. . 1 ...... Lispe sp...... 1 ...... Muscidae Gen. sp. ...1 . 1 ...... 1 ...... Pericoma sp...... 1 ...... 2 . 2 Pilaria sp...... 11...... Psychoda sp. ... 1 16...... 1 ...... 1 ....1 . 12 Psychodidae Gen. sp. ....11...... 2 . ..2 . 2 . 12...2 .. Tipula sp...... 2 ...... Wiedemannia sp...... 2 ......